The present invention relates generally to compositions that enhance the in vivo oxygenating properties of hemoglobin products. More particularly, the present invention relates to time-controlled superoxygenating compositions that comprise hemoglobin colloid and guanosine 3xe2x80x2:5xe2x80x2-cyclic monophosphate (cyclic GMP) generating compounds, and to methods for treatment of diseases or medical conditions which utilize the time-controlled superoxygenating compositions as biocolloids, i.e. hemodiluents, blood substitutes, plasma expanders, or resuscitative fluids.
There are many medical conditions, for example hemorrhagic hypotension and anaphylactic shock, in which significant blood loss and/or hypotension (abnormally low blood pressure) occur leading to reduced tissue oxygenation. For patients with such medical conditions, it is desirable and often critical for their survival to stabilize their blood pressure and to increase the amount of oxygen provided to body tissues by their circulatory systems.
Considerable effort has therefore been expended in developing colloidal substances which may be used as resuscitation fluids and/or blood plasma expanders for stabilizing blood pressure by hemodilution (i.e., increasing blood plasma volume) and which are capable of carrying and delivering oxygen to bodily tissues. The costs, risks (including contamination with disease-causing viruses) and histocompatibility requirements associated with the transfusion of whole blood or blood fractions have stimulated researchers to develop alternate oxygen-carrying substances.
Hemoglobin, the natural respiratory protein of erythrocyte which carries oxygen to body tissues from the lungs, is a potential alternate oxygen-carrying biocolloid. Erythrocytes contain approximately 34 grams of hemoglobin per 100 ml of red cells.
Hemodilution experiments with hemoglobin have revealed that unlike hemodilution with albumin, hemodilution with hemoglobin does not augment cardiac output (whole-body blood flow). During hemodilution with inert colloids such as albumin, whole-body blood flow increases inversely proportional to the level of hemodilution. In other words, an ≈50% hemodilution (i.e., an ≈50% decrease in blood red cell mass) increases blood flow ≈100%, and so on. The physical basis of the hematocrit-blood flow inverse relationship is analogous to viscosity-flow mechanics. In other words, decreases in hematocrit cause decreases in blood viscosity which results in increases in blood flow. Hemoglobin, on the other hand, is not inert. Hemodilution with hemoglobin causes vasoconstriction which results in smaller diameter blood vessels. Therefore, even though blood viscosity is decreased during hemodilution with hemoglobin, the smaller diameter blood vessels resist increases in blood flow. This is analogous to direct mechanical relationship between flow and tube diameter, i.e., decreased diameter results in decreased flow. Whole-body flow (cardiac output) is not increased during hemodilution with hemoglobin because flow of the less viscous blood is opposed by the hemoglobin-mediated decrease in blood vessel diameter.
Increased cardiac output is desirable to increase oxygen delivery to the tissues. Examples of publications describing the lack of increased cardiac output during hemodilution with natural (unmodified) hemoglobin are Sunder-Plassmann et al., Eur. J. Int. Care Med. 1, 37-42 (1975) and Moss et al., Surg. Gyn. Ob., 142, 357-362 (1976).
Hemodilution with modified hemoglobin that has been polymerized also has failed to increase cardiac output. See Vlahakes et al., J. Thorac. Cardiovasc. Surg., 100, 379-388 (1990) which describes the hemodilution of sheep with polymerized bovine hemoglobin prepared by Biopure Corporation; Rausch et al., supra (assigned to Biopure Corporation), which describes similar experiments; and Gould et al., Ann. Surg., 211, 394-398 (1990) and Hobbhahn et al., Acta Anaesthesiol. Scand., 29, 537-543 (1985) which describe hemodiluting baboons and dogs, respectively, with polymerized human hemoglobin solutions. Thus, hemodilution with both natural and modified hemoglobin has failed to increase cardiac output.
Because cardiac output does not increase upon dilution of the blood with hemoglobin, body tissues are required, as one compensatory mechanism, to extract more oxygen from the diluted blood to prevent tissue damage from hypoxia. However, such compensatory mechanisms have real limits in vivo. The heart, for instance, normally functions at about 95% maximal oxygen extraction levels and thus is only capable of increasing oxygen extraction about 5% (assuming 100% efficiency is possible). In the case of hemodilution with albumin, even though arterial oxygen content is decreased, oxygen delivery is essentially maintained at baseline levels because of low viscosity blood that causes flow (cardiac output) to increase proportionally. Therefore, hemodilution with hemoglobin offers no physiological or clinical advantage over hemodiluting with albumin. In fact, oxygen delivery is suboptimal during hemodilution with hemoglobin. This leads us to the central idea of the invention. If cardiac output or whole-body flow were allowed to increase as blood was diluted with hemoglobin then oxygen delivery would be clinically superior to hemodilution with albumin because of the added arterial oxygen content provided by the plasma hemoglobin colloid.
The mechanism of unchanged cardiac output during hemodilution with hemoglobin may be caused by an inactivation of endogenous nitric oxide (NO), also called endothelium-derived relaxing factor (EDRF), which is an important regulator of blood vessel diameter. For nearly 100 hundred years, it has been known that hemoglobin inactivates NO that has been diffused to the blood via the lungs. Only recently has it been discovered that NO also forms biochemically in vivo. There is, however, uncertainty as to whether oxyHb directly reacts with NO or whether an indirect oxyHb-mediated product such as superoxide is responsible. An overview of the possible molecular interactions of oxyHb with NO and relevant compounds is give below.
Initially, the reaction equations between NO and oxyHb/deoxyHb are simple yielding
oxyHb+NOxe2x86x92metHb+NO3xe2x88x92
deoxyHb+NOxe2x86x92NOHb 
where metHb is ferric(Fe3+)hemoglobin, NO3xe2x88x92 is nitrate and NOHb is nitrosylhemoglobin. However, NO3xe2x88x92 can react with deoxyHb to yield:
2deoxyHb+NO3xe2x88x92+H2Oxe2x86x922metHb+NO2xe2x88x92+2OHxe2x88x92
and nitrite (NO2xe2x88x92) can react with oxyHb or deoxyHb to yield:
2oxyHb+NO2xe2x88x92+H2Oxe2x86x922metHb+NO3xe2x88x92+2OH2xe2x88x92
oxyHb+NO2xe2x88x92+2H+xe2x86x92metHb+NO2+H2O2 
deoxyHb+NO2xe2x88x92+H2Oxc2xd[metHb+NO]+xc2xd[NOHb]+2OHxe2x88x92
where NO2 is nitrogen dioxide and H2O2 is hydrogen peroxide. The free energy of activation (xcex94G) of the last equation is rather low (xe2x88x9221.23 kJ/mole) and therefore metHb . . . NO can be reduced to deoxyHb. However, in vivo experiments have shown that nitrite exposure results in about an equal amount of NOHb and metHb formed in the blood. Furthermore, the xcex94G of equation 2 is low (xe2x88x9246.02 Kj/mole) and in the presence of O2 will proceed according to the xcex94G of equation 1(≈xe2x88x92170 Kj/mole). Finally, metHb and H2O2 can react:
metHb+H2O2xe2x86x92ferrylhemoglobin+H2O 
to produce a spectrophotometrically detectable red compound known as ferryl(Fe++++)hemoglobin. In many of the above reactions, heme or chelatable iron can be substituted for Hb.
Under physiological conditions, i.e., in an oxygenated and heated aqueous with a pH of about 7.4, NO is rapidly converted to nitrogen dioxide:
2 NO+O2xe2x86x922 NO2 
Nitrogen dioxide is quite reactive, and in aqueous solution disproportionates to nitrate and nitrite as:
2NO2xe2x86x92N2O4+H2Oxe2x86x92NO3xe2x88x92+NO2xe2x88x92+2H+
The decomposition of NO can also occur via:
NO2+NOxe2x86x92N2O3+H2Oxe2x86x922NO2xe2x88x92+2H+
The abnormally rapid oxidation of oxyHb by NO is consistent with oxyHb serving as a superoxide (O2xe2x88x92) donor, where
oxyHb+NOxe2x86x92metHb+ONOOxe2x88x92xe2x86x92NO3xe2x88x92
or as recent studies propose, that NO is inactivated by other sources of O2xe2x88x92 yielding:
NO+O2xe2x88x92xe2x86x92ONOOxe2x88x92+H+xe2x86x92HO.+NO2xe2x86x92NO3xe2x88x92+H+
where ONOOxe2x88x92 is peroxynitrite. Hydroxyl radical (HO.) can further react with NO2xe2x88x92 to form NO2. Peroxynitrate (O2NOOxe2x88x92) and ONOOxe2x88x92 are also suspected intermediates in the autocatalytic oxidation of oxyHb by NO2 and NO, respectively.
Considering the autoxidation of oxyHb:
oxyHb⇄metHb+O2xe2x88x92
O2xe2x88x92+oxyHbxe2x86x92metHb+O2+H2O2 
These last two equations are slow (the rate constants are 4-6xc3x97103 Mxe2x88x921secxe2x88x921). A currently-popular hypothesis is that O2xe2x88x92 is converted in the presence of iron to the highly toxic HO. radical via the superoxide driven Fenton reaction:
O2xe2x88x92+Fe3+xe2x86x92Fe+++O2 
2O2xe2x88x92+2H+xe2x86x92H2O2+O2 
H2O2+Fe++xe2x86x92HO.+OHxe2x88x92+Fe3+
Whether oxyHb+H2O2 forms HO. has not been proven. Furthermore, ferric iron (Fe3+) is sparingly soluble under physiological conditions and, therefore, must be chelated to heme, ferritin, etc to remain in solution. As with metHb, heme-Fe3+ may also react with H2O2 to form the ferryl-heme radical (.Fe4+-heme).
Finally, rather than binding to heme, inactivation of endogenous NO may occur via its reaction with the thiol groups of oxyHb. Recently, it was shown that NO circulates in mammalian plasma primarily as an S-nitroso adduct on the thiol groups of serum albumin.
The biochemical pathway/effect of NO production and metabolism is believed to comprise the following, L-argininexe2x86x92NO synthasexe2x86x92NOxe2x86x92guanylate cyclasexe2x86x92cyclic GMPxe2x86x92decreased blood vessel diameter. Conceivably, hemoglobin could decrease blood vessel diameter by inactivating any point of the pathway. Furthermore, hemoglobin does not effect the diameter of veins and arteries equally. Equal effect on these two vascular systems is necessary to increase blood flow or cardiac output as observed during albumin hemodilution. Therefore, two mechanisms must be synchronized in order for cardiac output (and oxygenation) to be maximized during hemodilution with hemoglobin: 1) molecular intervention of in vivo hemoglobin chemistry and 2) hemodynamic responsiveness in venous and arterial circulations.
There thus continues to exist a need in the art for new methods, and compositions, useful for hemodilution with hemoglobin which increase cardiac output (oxygen delivery) and give this colloid a clinical advantage over non-oxygenated colloids such albumin.
The present invention provides therapeutic compositions for hemodilution with hemoglobin products that superaugment the oxygenating capacity of the circulatory system when using these products. The cardiac output-increasing compositions comprise hemoglobin and guanosine 3xe2x80x2:5xe2x80x2-cyclic monophosphate (cyclic GMP) generating compounds. Methods of treatment for diseases and medical conditions requiring/indicating use of a superoxygenating compositions as hemodiluents, blood substitutes, plasma expanders, or resuscitative fluids are described. For use in therapeutic compositions of the present invention, the hemoglobin products may be modified to prevent rapid clearance from the intravascular space in vivo. For example, the hemoglobin products may be cross-linked, chemically modified with compounds such as polyethylene glycol or encapsulated (for example, in liposomes, glucose polymers or gelatin).
Therapeutic compositions according to the invention were formulated to reverse the vasoconstriction of hemoglobin products and to thereby facilitate the flow-increasing effects of diluted blood (augmented cardiac output). The compositions comprise either hemoglobin and cyclic GMP generating compounds or hemoglobin chemically coupled to cyclic GMP-generating compounds. Hemoglobin and excess nitric oxide were discovered to exhibit a combined pharmacology resulting in increased cardiac output in mammals. See Rooney et al., FASEB Journal, 5(4), Abstract 158 (1991).
Appropriate cyclic GMP-generating compounds contemplated by the present invention include, for example, nitric oxide precursors which are endogenous to mammals (e.g., L-arginine, lysine, glutamate, ornithine) and engineered compounds which contain nitric oxide groups (e.g., the nitrovasodilators sodium nitroprusside, organic nitrates, S-nitrosothiols, sydnonimines and furoxans) or which cause the release of nitric oxide in vivo (e.g., the endothelium-dependent vasodilators acetylcholine, bradykinin, adenine nucleotides and substance P) or any compound which may directly or indirectly activate guanylate cyclase (dihydropyridines and related nitrovasodilator-dihydropyridine hybrid structures).
Cyclic GMP-generating compounds may be coupled to hemoglobin by chemical processes which selectively attach the compounds to reactive amino (e.g., lysine residues) carboxyl (e.g., glutamate residues) or previously thiolated-amino groups on the hemoglobin surface. Native thiol and disulfide groups of hemoglobin that play an important structural or functional role should be avoided.
Typically, a bifunctional reagent such as an imidoester may be used to couple a cyclic GMP-generating compound to reactive hemoglobin groups. In some cases, a group on the cyclic GMP-generating compound may be activated prior to incubation with hemoglobin. For example, a carboxyl group on the cyclic GMP generating compound may be activated with a carbodi-imide for coupling to amino groups on the hemoglobin surface. Appropriate coupling reactions may involve other reagents, for example, sodium cyanoborohydride, carboxi-imides, succinic anhydride, thiols, N-hydroxysuccinimide and dithiothreitol.
The chemical coupling of the hemoglobin and the cyclic GMP-generating compound may involve a reversible or an irreversible bond. See, for example, the coupling reactions in Carlsson et al., Biochem. J., 173, 723-737 (1978) and Martin et al., J. Biol. Chem. 249, 286-288 (1981). It may be useful to react the xcex2 or xcex1 chain lysines or the amino terminal residues of hemoglobin with agents that increase relative oxygen dissociation [see Chatterjee et al., J. Biol. Chem., 261, 9929-9937 (1986)] before coupling the hemoglobin to the cyclic GMP-generating compounds.
The therapeutic hemoglobin compositions of the present invention may be described as xe2x80x9cblood component substitutes.xe2x80x9d In addition to hemoglobin and cyclic GMP-generating compounds, the compositions may comprise physiologically acceptable plasma substitutes. Suitable plasma substitutes are linear polysaccharides (e.g., dextrans, gum arabic pectins, balanced fluid gelatin, and hydroxyethyl starch), polymeric substitutes (e.g., polyethylene oxide, polyacrylamide, polyvinyl pyrrolidone, polyvinyl alcohol, ethylene oxide-propylene glycol condensate), aqueous solutions (e.g., Lactated Ringers and saline), coacervates (composed of fatty acids, phospholipids, glycerates or cholesterol, for example) and colloidal substitutes (e.g., albumin).
The therapeutic compositions according to the present invention are useful for treatment of diseases or medical conditions in which intravascular or intraosseous administration of a resuscitative fluid or blood plasma expander is indicated/required. Resuscitative fluids and blood plasma expanders are required for treatment of diseases and medical conditions in which there is significant blood loss, hypotension and/or a need to maximize the availability of oxygen to the body tissues. Examples of such diseases and medical conditions are hemorrhagic hypotension, septic shock, cardio-pulmonary bypass, sickle cell and neoplastic anemias, plasma and extracellular fluid loss from burns, stroke, angioplasty, cardioplegia, radiation therapy, acute myocardial infarction, and both routine and lengthy surgical procedures.
Methods of treating such diseases and medical condition according to the present invention comprise the step of hemodiluting a mammal with a pharmaceutically effective amount of a hemoglobin composition according to the present invention. Hemodilution with the compositions is performed in a manner conventional in the art, for example, as described in Messmer et al., Prog. Surg., 13, 208-245 (1974). The present invention also contemplates that cyclic GMP generating compounds may simply be infused peri-hemodilution with a hemoglobin product. The methods of treatment of the invention are particularly useful in treating humans.
The contents of this specification are submitted as sufficient objective factual evidence that the invention claimed is not prima facia obvious under 35 U.S.C xc2xa7 103 and that the invention claimed could not have been expected to be achieved by one of ordinary skill since the art was not of public record at the time the invention was made.
The prior art only demonstrated that hemodilution, i.e. substitution of red cell mass, with hemoglobin failed to provide increased cardiac output or oxygen delivery. Exactly how this happens or the expected methods of reversing this result were not known in the art at the time the invention was made. The prior art did not know that hemoglobin-based material selectively inhibits vasomotor activity in arteries that is different from the inhibited vasomotor activity by hemoglobin-based material in veins. It was not known by anyone skilled in the art, except the applicant, that coupling of hemoglobin and nitric oxide groups (nitrovasodilators) and particularly hemoglobin and NO from Na nitroprusside (SNP) would increase cardiac output. The prior art knew for over 100 years that hemoglobin-based material binds nitric oxide, that hemoglobin-based material is a potential biocolloid, and that SNP is a vasodilator, however, the invention claimed was not prima facia obvious and was pure luck. In fact, the applicant expected that the NO donor used from SNP would not work since, in control animals or patients, SNP dilates arteries and veins equally, decreases blood pressure and rarely increases cardiac output unless heart rate increases. Therefore, the unexpected result was that SNP can not dilate veins in the presence of hemoglobin-based material thereby not affecting preload. This result discovered that hemoglobin does not effect arteries and veins equally. Blood pressure was also little affected, another unexpected result. Because of these unexpected results, stroke volume (i.e. cardiac output) was allowed to increase inversely proportional to the reduced viscosity component of afterload.
This invention will not work with every NO donor. For example, nitroglycerin and cyclic GMP analogues, in their present commercial formulation, will dilate both veins and arteries, with or without hemoglobin-hemodilution. There is no increase in cardiac output because both preload and afterload are reduced. This is further evidence that hemoglobin-based material affects arteries and veins by different mechanisms and that one skilled in the art could not expect this result, or, one skilled in the art could not expect that the invention claimed could be achieved.
The invention uses a series of related dihydropyridines to selectively provide various time-controlled oxygen deliveries. These compounds are classically slow channel calcium antagonists that preferentially dilate arteries to lower blood pressure over specific time periods. These dihydropyridines are structurally similar (The Merck Index). This class of slow channel calcium antagonist dihydropyridines are recognized to have the same pharmacological action (Goodman and Gilman""s The Pharmacological Basis of Therapeutics, Hardman et al., eds, Ninth Edition, table 32-2. In the absence of hemoglobin products, the dihydropyridine compounds generate cyclic GMP with increases of 20-70% from baseline levels. In the presence of hemoglobin products, the dihydropyridine compounds provide hemodynamics similar to those achieved with SNP, i.e. little effect on blood pressure and preload but appropriate reversal of hemoglobin antagonism in arteries. The fact that these compounds are unexpectedly similar to SNP in dilating arteries preferentially over veins suggests that hemoglobin-based material probably antagonizes the guanylate cyclase enzyme rather than scavenging NO directly or antagonizing the NO synthase enzyme. Oxygen delivery, therefore, is similar to SNP but has the additional advantage of being time-controlled.
The invention uses cyclic GMP generating compounds as the principle means of increasing oxygen delivery with any hemoglobin-based material. The evidence from the SNP and dihydropyridine studies and other studies of record presented in this communication clearly demonstrate that hemoglobin-based material inactivates or antagonizes more than one N)xe2x86x92cyclic GMP mechanism, i.e. effects on NOxe2x86x92cGMP mechanisms in arteries are not expected to be physiologically equivalent to those in veins. Furthermore, the reversal of hemoglobin inactivation or antagonism is dependent on more than one NOxe2x86x92cyclic GMP mechanism, i.e. reversal of NOxe2x86x92cGMP antagonism in arteries is not expected to be physiologically equivalent to that in veins. Therefore, the invention selectively acts on those NOxe2x86x92cGMP mechanisms to decrease afterload while having little effect on NOxe2x86x92cGMP mechanisms that control preload.
In summary, the specification is submitted as sufficient objective factual evidence, with unexpected results, that the invention claimed is not prima facia obvious under 35 U.S.C xc2xa7 103 and that the invention claimed could not have been expected to be achieved by one of ordinary skill in the art since the art or knowledge of the selective vasoactive mechanisms of hemoglobin-based material and claimed compounds required for control of these mechanisms was not known at the time the invention was made.